Fig 2: Compromised Affinity Maturation in the Absence of PD-1-PD-L1 Interactions between GC Tfh and B CellsMixed BM chimeras were constructed using 50% CD45.2 Cd274+/+ or Cd274-/- BM cells and 50% CD45.1 BM cells. Chimeric mice were immunized with NP-KLH.(A) Gating strategies for total B cells, plasma cells, GC B cells, NP-specific GC B cells, and memory B cells (top row) and for CD45.1 and CD45.2 cells within each indicated population (bottom row). The example is from Cd274-/-:CD45.1 chimera.(B) Scatterplots of competitive competencies of CD45.2 cells in the two sets of mixed chimeras contributing to GC B cells, NP-specific GC B cells, NP-specific memory B cells and plasma cells, and BM plasma cells at day 8, day 13, or day 21 after immunization. The competitive competency is defined as the CD45.2/CD45.1 ratio in the indicated compartment normalized against the same ratio in the total B cell compartment or the GC compartment in the case of NP-specific GC cells. Data are pooled from three independent experiments involving 3–4 mice per group per experiment. Each symbol denotes one mouse. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.(C) Total mutation rates and frequencies of affinity-enhancing W33L mutation in NP-specific, VH186.2-carrying GC B cells isolated on day 13 after NP-KLH immunization. Numbers in the center of the pie chart indicate total numbers of clones analyzed, and numbers of W33L+ or W33L- cells are indicated. Numbers of total mutation are presented as mean ± SEM. Frequencies of W33L mutations were compared by the Fisher’s exact test. Data are pooled from three independent experiments, each involving 3–4 mice for each group.

Fig 3: Inflammasome-dependent T cell exhaustion in Tmem176b-/- mice infected with MHV-A59.(A) Survival of MHV-A59–infected WT and Tmem176b-/- mice left untreated. In another group, infected WT mice were adoptively transferred with splenic DCs from infected Tmem176b-/- animals, and Tmem176b-/- mice were adoptively transferred with CD8+ T cells from infected WT animals. *P < 0.05; ***P < 0.001, log-rank (Mantel-Cox) test. (B) Percentage of MHV-A59–specific CD8-dependent in vivo lysis was calculated with the formula described in Materials and Methods. Twelve WT, 16 Tmem176b-/-, and 5 Tmem176b-/-Casp1-/- mice were studied at 5 dpi. ***P < 0.001; ****P < 0.0001, one-way ANOVA test. (C) Representative histograms of the experiments shown in (B). (D) Percentage of liver-infiltrating TOX+ TCF-1+ within PD-1+ CD44+ cells, gated in CD8+ MHV-A59–specific T cells. Animals from two independent experiments were analyzed. *P < 0.05, one-way ANOVA test. (E) Representative dot plots of the animals analyzed in (D). (F) Survival of Tmem176b-/- mice infected with MHV-A59 and treated with control IgG or anti–PD-1 antibody. ****P < 0.0001, log-rank (Mantel-Cox) test. (G) Viral load in the liver at 5 dpi with MHV-A59 in Tmem176b-/- mice treated with control IgG or anti–PD-1 antibodies. *P < 0.05, Mann-Whitney test. (H) Survival of WT mice infected with MHV-A59 and treated with control IgG or anti–PD-1 antibody. ns, nonsignificant. Log-rank (Mantel-Cox) test. (I) Viral load in the liver at 5 dpi with MHV-A59 in WT mice treated with control IgG or anti–PD-1 antibodies. Mann-Whitney test.

Fig 4: Recombinant IFNß promotes apoptosis, activation, inhibitory receptor expression, and dysregulated expression of the CAR.a Annexin V expression on CD8 CAR T cells or untransduced (UTD) T cells cultured in IL2 (50 U/mL) in the presence or absence of recombinant murine IFNß and 1µM Ruxolitinib (JAK1/2 inhibitor) for 48 h. b Annexin V expression on CD8 CAR T cells cocultured with B16EGFRvIII cells at an E:T ratio of 1:5 in the presence or absence of recombinant murine IFNß and/or 1µM Ruxolitinib. Fas (c) or cleaved caspase (d) expression on CD8 CAR T cells or UTD cells cultured in IL2 with IFNß. e Median fluorescence intensity (MFI) of CAR expression measured using Biotin-Protein L and streptavidin- PE (Protein L + SA PE) compared to Annexin V on CAR T cells cultured in IL2 with or without IFNß (10 ng/mL). CAR expression on CD8 CAR T cells cultured in IFNß for 48 h in the absence (f) or presence of target tumor cells (g). Representative (h) and mean (i) expression of CD25 and CD69 for CAR T cells cultured in IL2 with IFNß. Statistical comparisons are indicated for CD25- CD69- populations in (i). j Co-expression of the CAR and activation markers on CD8 CAR T cells. k Mean expression of PD1, LAG3, TIM3 on CD8 CAR T or UTD cells cultured in IL2 or in the presence of B16EGFRvIII cells (E:T 1:5). l Fold change in inhibitory receptor and CAR expression is shown for cells grown in IL2 with additional recombinant IFNß relative to culture in IL2 alone. m WT or transgenic IFNAR1 KO CAR T cells were cocultured with B16EGFRvIII (E:T ratio of 1:5) which were mock infected or infected with VSVmIFNß 6 h prior to coculture. Surface CAR expression is represented as fold change relative to coculture with mock infected tumor cells. n Inhibitory receptor expression was quantified on WT or IFNAR1 KO CD8 CAR T cultured as in (m). Data are representative of two independent experiments (panels a–l). Experiments in panels (m, n) were performed once. Means ± SD of n = 4 (panel a) or n = 3 (panels b–d, f, g, i, k, m, n) technical replicates are shown. P-values were determined using a two-way ANOVA with a Tukey multiple comparisons post-test (a–d, f, g, i). Statistical significance set at p < 0.05, ns > 0.05. Source data are provided in the Source Data File.

Fig 5: Recombinant IFNß promotes human CAR T cell apoptosis and inhibitory receptor expression.CAR and untransduced (UTD) T cells from three healthy donors were cultured in the absence or presence of human IFNß (0, 0.1, 1, or 10 ng/mL) for 48 h and analyzed for the expression of (a) cleaved caspase 3, (b) TIM3, (c) LAG3, and (d) PD1. e Average expression of individual and combinations of inhibitory receptors for three donors. Data are representative of two independent experiments. Data points indicate the average of two technical replicates for each donor and P-values were determined for all three donors using a two-way repeated measures ANOVA with a Tukey multiple comparisons post-test. Source data are provided in the Source Data File.